We propose to develop an alternative approach to address and treat HBV infection with targeted rare-cutting endonucleases. We will use zinc finger nucleases (ZFNs) to induce double-strand breaks within specific HBV sequences upon AAV-mediated ZFN delivery. We hope to achieve virologic cure of infected cells by promoting cellular degradation of cccDNA present in infected hepatocytes upon linearization or inactivating HBV genes within the episomal cccDNA. The repair mechanism for double-strand breaks is error prone and should result in disruption of genes around the cleaved target sequence. This proposal will study the efficacy and safety of our approach in the highly relevant humanized uPA-SCID mouse model of HBV infection. Our preliminary data shows that we can successfully mutate targeted HBV genes and inhibit HBV replication in an in vitro cell culture model of HBV using AAV-delivered ZFNs. It also shows that our HBV-specific ZFNs cause minimal levels of off-target cleavage at similar sites within the human genome. We have established the uPA-SCID mouse model of HBV infection and have demonstrated efficient delivery of a fluorescent reporter gene to human hepatocytes in humanized mouse livers using AAV vectors. Our comprehensive preliminary data provides the basis for a pilot study of the antiviral efficacy of this therapeutic approach in vivo. The overall aim of this study is to provide proof of principle fr its use in treating chronic HBV infections.